The principal goal of this project is to characterize the mechanism of oxygenation by 4-methoxybenzoate monooxygenase (4MBMO). 4MBMO is a non- heme iron oxygenase with two components: an oxygenase protein, putidamonooxin (PMO) and an NADH-dependent oxide reductase, putidamonooxin reductase (PMOR). This enzyme has exceptional catalytic versatility that makes it well suited for characterizing the mechanism of oxygenation by the Fe-Rieske class of oxygenases. The long-term goals of the project include a structure-function analysis of the mechanism of the Fe-Rieske enzymes. In this proposal a series of physical-chemical experiments are proposed that focus on the oxygenation step. It is anticipated that this work will illuminate not only the mechanism of the Fe-Rieske enzymes, but contribute to our understanding of iron-oxygen chemistry. A variety of enzymes, including cytochrome P- 450's, utilize iron and exogenous reducing equivalents to activate oxygen for insertion into a C-H bond. Work on these systems should be aided by characterization of the Fe-Rieske enzymes. Characterization of the Fe-Rieske enzymes should also facilitate use of bacteria for biodegradation of xenobiotic chemicals. The specific aims of this work include 1. Rapid Reaction Studies of the kinetics of the reaction of reduced putidamonooxin (PMO, the oxygenase of the 4MBMO system) with O2 and substrate will be performed. These studies should provide a minimal kinetic mechanism for the oxygenative half-reaction; the first such description of an Fe-Rieske enzyme. 2. Kinetic Isotope Effects Steady state deuterium kinetic isotope effects (DV, and DV/Km) on demethylation of 4-methoxybenzoate will be measured to determine the extent of rate-limitation by cleavage of the C-H bond on the substrate. Characterization of the intrinsic (intramolecular) kinetic isotope effect will test the proposal that hydrogen abstraction participates in the oxygenation of alkyl moieties. 3. Thermodynamics of reduced putidamonooxin-substrate complex. Thermodynamic analysis of the reduced PMO-substrate complex and the oxidized PMO-product complex will complement to kinetic analysis of the oxygenation reaction. This work will test whether thermodynamic forces associated with substrate-binding facilitate electron transfer to oxygen. These measurements will also aid in characterizing the kinetics of substrate binding and product dissociation. 4. Reaction of PMO with H2O2 and other oxygen donors PMO will react with H2O2 and demethylate 4-methoxybenzoate. This exciting new observation provides a unique window onto the oxygenation reaction. Further characterization of this reaction (and testing other oxygen donors) will help describe the active-oxygen species in the Fe-Rieske systems.